Variants of pigment epithelium derived factor and uses thereof

ABSTRACT

The present invention provides anti-angiogenic variants of pigment epithelium derived factor (PEDF) comprising at least one altered phosphorylation site, polynucleotides encoding same and uses thereof. Particularly, the present invention provides variants of human PEDF comprising at least one amino acid substitution at serine residues (24), (114), and (227). The PEDF variants are potent anti-angiogenic factors, and thus useful in treating diseases or disorders associated with neovascularization.

FIELD OF THE INVENTION

The present invention provides anti-angiogenic variants of pigmentepithelium derived factor (PEDF) comprising an altered phosphorylationsite, polynucleotides encoding same and uses thereof. In particular, thevariants of the invention provide improved anti-angiogenic activitywhile being devoid of certain undesirable properties of PEDF obtainedfrom natural sources.

BACKGROUND OF THE INVENTION

Pigment epithelium derived factor (PEDF) was originally identified inconditioned medium of fetal human retinal pigment epithelium cellcultures. It shares sequence and structure homology to members of thesuperfamily of serine protease inhibitors (serpin), however, it does notserve as an inhibitor of any known protease activity.

PEDF was first described as a neurotrophic factor that induces aspecific neuronal phenotype in retinoblastoma cells (Steel, F. R. et al.Proc. Natl. Acad. Sci. U.S.A. 90: 1526-1530, 1993). The neurotrophicactivity of PEDF was also demonstrated by its ability to supportneuronal survival (Taniwaki, T. et al. J. Neurochem. 64: 2509-2517,1995), and its ability to protect neurons against neurotoxic effects.Structure-function studies have shown that this neurotrophic activity isexerted by the amino terminal segment (44-mer, amino acid residues78-121) of the human PEDF, and that its activity is mediated through a˜80 kDa membranal receptor, which is abundant in retinoblastoma cells,and in neural retinal cells.

Besides its neurotrophic activity, PEDF was further demonstrated to beone of the most potent natural inhibitors of angiogenesis (Dawson, D. W.et al. Science 285: 245-248, 1999). Thus, it was found that PEDFinhibits not only bFGF-induced migration of endothelial cells under invitro conditions, but also bFGF-induced neovascularization in anavascular rat cornea. Furthermore, addition of anti-PEDF antibodies(Abs) to rat corneas was found to stimulate the invasion of new vesselsinto these corneas, suggesting that PEDF plays a physiologicalregulatory role in retinal angiogenesis. PEDF was also shown to be avery potent inhibitor of neovascularization in a murine model ofischemia-induced retinopathy (Stellmach, V. V. et al. Proc. Natl. Acad.Sci. U.S.A. 98: 2593-2597, 2001). The anti-angiogenic activity of PEDFwas associated with endothelial cell apoptosis, probably by increasingFas ligand (FasL) mRNA and surface FasL in these cells.

It was recently reported that besides its expression in multiple sitesin the eye, PEDF is also present in human plasma, at a physiologicallyrelevant concentration (Petersen, S. V. et al. Biochem. J. 374: 199-266,2003). In the last decade several reports have described the possibilitythat protein kinases might function as a regulatory device not onlyintracellularly, but also in the cell exterior (Redegeld, F. A. et al.Trends Pharmacol. Sci. 20: 453-459, 1999). These reports described thepresence of membrane-bound ectoprotein kinases (on the outer cellsurface) and soluble secreted exoprotein kinases (detached from thecell). Additionally, it was shown that these ecto- or exoprotein kinasesdo have several substrates in the circulating blood including thecoagulation cofactors Va and VIII as well as vitronectin. The mainprotein kinases that seem to exert exokinase activity are protein kinaseA (PKA) and protein kinase CK2 (CK2). For example it was shown thatvitronectin is phosphorylated by PKA and this phosphorylation modulateits interaction with PAI-1. In addition, phosphorylation by CK2 changesintracellular signaling by vitronectin, indicating that both PKA and CK2play an important regulatory role in the circulating blood.

U.S. Pat. No. 5,840,686 to Chader et al., discloses nucleic acids thatencode PEDF and a truncated PEDF, the equivalent proteins, and methodsfor producing recombinant PEDF and the truncated PEDF. U.S. Pat. No.5,840,686 claims a method of prolonging neuronal cell survival and amethod for inhibiting glial cell proliferation comprising administeringrecombinant PEDF. U.S. Pat. No. 6,319,687 to Chader et al., claims arecombinant PEDF protein (418 amino acids) and truncated forms of PEDFhaving neurotrophic as well as gliastatic activity.

PCT Application WO 01/58494 claims a method of treating anocular-related disease in an animal. The method comprises expression ofan angiogenesis inhibitor and a neurotrophic agent in an ocular cellusing an expression vector that contains the nucleotide sequence forthese factors. A preferred angiogenesis inhibitor is PEDF, which isknown to exert both anti-angiogenic and neurotrophic activities.

U.S. Pat. No. 6,451,763 to Tombran-Tink et al., discloses thepurification of PEDF from culture medium of human retinal pigmentepithelial cells and claims methods of treating retinal diseasescomprising administering PEDF to subjects in need thereof. It is alsodisclosed that in addition to retinal pigment epithelial cells, PEDF maybe isolated from the vitreous humor of human, bovine, monkey and otherprimates. Since PEDF is abundant in the vitreous humor and since thevitreous humor is easily removed from the eyecup, the vitreous humor wassuggested to be the easiest source from which PEDF can be isolated.

U.S. Pat. No. 6,797,691 to Bouck et al., discloses methods of inducingdifferentiation and slowing the growth of a neuroblastoma cellcomprising administering PEDF to the cell.

International Patent Application WO 03/059248 discloses that PEDF ispresent in human plasma at physiologically relevant concentrations andexhibits potent anti-angiogenic and neurotrophic activities.

The inventors of the present application have shown that mutations ofthe phosphorylation sites of PEDF affected its anti-angiogenic andneurotrophic activities (Seger et al., The Weizmann Institute BioScienceOpen Day May 17, 2004; Seger et al., FEBS Lecture Course on CellularSignaling, May 21-27, 2004; Seger et al., Blood, in press, 2004), buthave not disclosed the utility of the present variants.

It would be highly advantageous to have PEDF variants having greaterselectivity in terms of their anti-angiogenic and neurotrophic activitythan native or wild-type PEDF.

SUMMARY OF THE INVENTION

The present invention provides anti-angiogenic variants of pigmentepithelium derived factor (PEDF) of SEQ ID NO:1 comprising at least onealtered phosphorylation site. The present invention further providespolynucleotides encoding the PEDF variants of the invention, expressionvectors comprising same, and methods of treating diseases or disordersassociated with neovascularization.

The present invention is based in part on the identification of PEDFphosphorylation sites. It is now disclosed, for the first time, thathuman PEDF undergoes casein kinase 2 (CK2) phosphorylation on the serineresidues 24 and 114 and protein kinase A (PKA) phosphorylation on theserine residue 227.

Unexpectedly, substitution of the serine residues 24 and 114 by glutamicacid resulted in the production of a human PEDF variant, i.e., S24,114E, having highly potent anti-angiogenic activity but essentiallydevoid of neurotrophic activity. The anti-angiogenic activity of theS24, 114E variant was higher than that obtained by the wild-typerecombinant human PEDF or by the naturally occurring human PEDF, e.g.,human plasma PEDF.

The PEDF variants are, therefore, very useful in treating diseases ordisorders associated with neovascularization. It should be understoodthat the production of a PEDF variant is advantageous as largequantities of homogeneous anti-angiogenic PEDF are obtained.Additionally, the PEDF variants are essentially free from anydisease-causing agents or any other undesirable proteins, which mayaccompany PEDF obtained from natural sources. It should also beappreciated that a PEDF variant having high anti-angiogenic activity butlower neurotrophic activity or even essentially devoid of neurotrophicactivity compared to the recombinant wild-type PEDF is highlyadvantageous in treating diseases associated with neovascularization,particularly malignant conditions, where the neurotrophic activity ofPEDF is undesirable.

According to one aspect, the present invention provides ananti-angiogenic variant of PEDF, an analog, or a fusion protein thereofcomprising the amino acid sequence of SEQ ID NO:1 or a fragment thereofcomprising at least one altered phosphorylation site.

According to some embodiments, the variant of PEDF, analog, fusionprotein, or fragment thereof has lower neurotrophic activity compared torecombinant wild-type PEDF. According to some preferred embodiments, thevariant of PEDF, analog, fusion protein, or fragment thereof isessentially devoid of neurotrophic activity.

According to additional embodiments, the at least one alteredphosphorylation site of the PEDF variant, fragment, analog, or fusionprotein thereof is selected from the group consisting of serine residues24, 114, and 227. According to some embodiments, the PEDF variant,analog, or fusion protein thereof comprises an amino acid sequenceselected from any one of SEQ ID NO:2 to SEQ ID NO:13 or a fragmentthereof. According to other embodiments, the phosphorylation site issubstituted by an amino acid selected from polar neutral amino acids,non-polar amino acids, negatively charged amino acids, and positivelycharged amino acids. According to currently preferred embodiments, theserine residue is substituted by a negatively charged amino acid,preferably by a glutamic acid. According to some embodiments, thepresent invention provides a PEDF variant, analog, or a fusion proteinthereof, wherein the serine residue at position 24 is substituted by aglutamic acid, thus resulting in a PEDF variant of SEQ ID NO:2 or afragment thereof. According to additional embodiments, the presentinvention provides a PEDF variant, analog, or a fusion protein thereof,wherein the serine residue at position 114 is substituted by a glutamicacid, thus resulting in a PEDF variant of SEQ ID NO:5 or a fragmentthereof. According to yet other embodiments, the present inventionprovides a PEDF variant, analog, or a fusion protein thereof, whereinthe serine residues at position 24 and 114 are substituted by glutamicacids, thus resulting in a PEDF variant of SEQ ID NO:8 or a fragmentthereof.

According to some other embodiments, the serine residue of PEDF isaltered by a chemical modification. Chemical modifications of an aminoacid are well known in the art and include, but are not limited to,glycosylation, oxidation, permanent phosphorylation, reduction,myristylation, sulfation, acylation, acetylation, ADP-ribosylation,amidation, hydroxylation, iodination, methylation, and derivatization byprotecting/blocking groups. Preferably, the chemical modification ispermanent phosphorylation

According to another aspect, the present invention provides an isolatedpolynucleotide sequence encoding an anti-angiogenic variant of PEDF,analog, or a fusion protein thereof, the anti-angiogenic variant ofPEDF, analog, or fusion protein thereof comprising the amino acidsequence of SEQ ID NO:1 or a fragment thereof comprising at least onealtered phosphorylation site.

According to some embodiments, the variant of PEDF, fragment, analog, orfusion protein thereof encoded by the isolated polynucleotide comprisesat least one altered phosphorylation site, wherein the at least onealtered phosphorylation site is selected from the group consisting ofserine residues 24, 114, and 227. According to some embodiments, theisolated polynucleotide encodes a PEDF variant, analog, or a fusionprotein thereof, the PEDF variant, analog, or fusion protein thereofcomprising the amino acid sequence selected from any one of SEQ ID NO:2to SEQ ID NO:13 or a fragment thereof. According to other embodiments,the serine residue is substituted to an amino acid other than serine.According to some embodiments, the isolated polynucleotide sequence isthus selected from any one of SEQ ID NO:15 to SEQ ID NO:22. According tocurrently preferred embodiments, the serine residue is substituted by anegatively charged amino acid, preferably by a glutamic acid. Accordingto additional embodiments, the isolated polynucleotide sequence isselected from any one of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, andSEQ ID NO:22 or a fragment thereof, which correspond to serinesubstitution by glutamic acid at position 24, 114, both 24 and 114, and227, respectively.

According to a further aspect, the present invention provides anexpression vector comprising an isolated polynucleotide sequenceencoding an anti-angiogenic variant of PEDF, analog, or a fusion proteinthereof, the anti-angiogenic variant of PEDF, analog, or fusion proteinthereof comprising the amino acid sequence of SEQ ID NO:1 or a fragmentthereof comprising at least one altered phosphorylation site.

According to yet another aspect, the present invention provides a hostcell transfected with an expression vector according to the principlesof the present invention.

According to a further aspect, the present invention provides apharmaceutical composition comprising as an active ingredient ananti-angiogenic variant of PEDF, analog, or a fusion protein thereof,the PEDF variant, analog or fusion protein thereof comprising the aminoacid sequence of SEQ ID NO:1 or a fragment thereof comprising at leastone altered phosphorylation site according to the principles of thepresent invention, and a pharmaceutically acceptable carrier. Thepresent invention further provides pharmaceutical compositionscomprising as an active ingredient an isolated polynucleotide encodingan anti-angiogenic variant of PEDF, fragment, analog, or a fusionprotein thereof according to the principles of the invention, and apharmaceutically acceptable carrier.

According to another aspect, the present invention providespharmaceutical compositions comprising as an active ingredient anexpression vector comprising an isolated polynucleotide encoding ananti-angiogenic variant of PEDF, fragment, analog or fusion proteinthereof according to the principles of the invention. According to someother embodiments, the pharmaceutical compositions of the inventioncomprise as an active ingredient a host cell transfected with anexpression vector comprising an isolated polynucleotide encoding ananti-angiogenic variant of PEDF, fragment, analog, or fusion proteinthereof according to the principles of the invention.

According to a further aspect, the present invention provides a methodfor treating a disease or disorder associated with neovascularization ina subject, the method comprising administering to the subject in needthereof a therapeutically effective amount of a pharmaceuticalcomposition according to the principles of the invention.

According to some embodiments, the disease or disorder associated withneovascularization is selected from malignant and metastatic conditions,ocular disorders, and disorders treated with anti-angiogenic factors.

According to other embodiments, the disease or disorder associated withneovascularization is selected from the group consisting of sarcoma,carcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor leiomydsarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumorcervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, Kaposi's sarcoma,pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma,menangioma, melanoma, neuroblastoma, retinoblastoma, neovascularglaucoma, diabetic retinopathy, retinoblastoma, retrolentalfibroplasias, uveitis, retinopathy of prematurity, macular degeneration,corneal graft neovascularization, retinal tumors, choroidal tumors,hemangioma, arthritis, psoriasis, angiofibroma, atherosclerotic plaques,hemophilic joints, and hypertrophic scars.

According to another aspect, the present invention provides a method fortreating a neurodegenerative condition in a subject comprisingadministering to the subject in need thereof a therapeutically effectiveamount of a pharmaceutical composition according to the principles ofthe invention and a pharmaceutically acceptable carrier.

According to some embodiments, the neurodegenerative condition isselected from the group consisting of neurodegenerative diseases andother insults of the CNS (brain and retina), which are typified by deathof neurons and overpopulation by glial cells (gliosis).

These and other embodiments of the present invention will be betterunderstood in relation to the figures, description, examples and claimsthat follow.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-D show that PEDF in plasma is a phosphoprotein. A, recombinantPEDF (rPEDF), plasma PEDF (plPEDF), active (phosphorylated) ERK,phosphorylated α-casein (phos cas) and dephosphorylated α-casein (dephoscas) were subjected to gel electrophoresis and immunoblotting withanti-phospho Ser, Thr, or Tyr Abs in the presence or absence of theappropriate phosphorylated amino acid. As a control, the samples wereblotted with anti-PEDF, anti-phosphorylated ERK (αpERK) and anti-generalERK (αgERK) Abs, or stained with gel code for the α-casein. B, rPEDF orplPEDF were incubated in the absence or presence of alkaline phosphatase(APase) conjugated to beads. The samples were subjected to in vitro CK2or PKA phosphorylation. Phosphorylated products were analyzed byautoradiography (Auto, upper panel) or by immunoblotting with anti-PEDFAb (lower panel). C, Quantitative analysis of the experiment depicted inFIG. 1B. D, Plasma PEDF was subjected to alkaline phosphatase treatmentand incubated with fresh human plasma and [γ³²P]-ATP in the presence orabsence of PKA inhibitor (PKI) or heparin. Control samples weresubjected to in vitro CK2 or PKA phosphorylation. Vn—plasma vitronectin.

FIGS. 2A-E show the CK2 and PKA phosphorylation of PEDF in vitro. A,rPEDF and plPEDF were incubated with CK2, [γ³²P]-ATP and increasingconcentrations of poly-L-lysine (PLL). The samples were subjected to gelelectrophoresis. The gel was stained with Coomassie blue (Coom, lowerpanel), and subjected to autoradiography (Auto, upper panel). B, rPEDFand plPEDF were incubated with CK2, [γ³²P]-ATP, poly-L-lysine, andincreasing concentrations of heparin (Hep). Phosphorylation was detectedby autoradiography. C, rPEDF and plPEDF were incubated with thecatalytic subunit of PKA, heparin, and [γ³²P]-ATP. Phosphorylation wasdetected by autoradiography. D, rPEDF was digested with trypsin for theindicated time periods. The samples were subjected to gelelectrophoresis followed by silver staining of the gel (left panel).rPEDF was radioactively phosphorylated by CK2 and then subjected totrypsin digestion and to gel electrophoresis followed byautoradiography. E, Schematic representation of PEDF showing the CK2 andPKA phosphorylation sites and the tryptic peptides revealed by massspectrometry and N-terminus sequence analysis.

FIGS. 3A-C show the identification of CK2 and PKA phosphorylation sitesof PEDF by site directed mutagenesis. A, rPEDF and rPEDF variants wereradioactively phosphorylated by CK2. The samples were subjected to gelelectrophoresis. The gel was stained with Coomassie blue (Coom, lowerpanel), and subjected to autoradiography (Auto, upper panel). B, rPEDFand rPEDF variants were radioactively phosphorylated by PKA. Sampleswere subjected to gel electrophoresis. The gel was stained withCoomassie blue (Coom, lower panel), and subjected to autoradiography(Auto, upper panel). C, Quantitative analysis of the autoradiogramdepicted in FIGS. 3A and B.

FIGS. 4A-E show the effect of rPEDF, plPEDF and the various rPEDFvariants on ERK/MAPK activation in HUVEC. A, Endothelial cells (HUVEC)were stimulated with 10 and 100 nM of rPEDF. Cytosolic extracts weresubjected to immunoblotting with anti-phosphorylated ERK (αpERK, upperpanel) or anti-general ERK (αgERK, lower panel) Abs. B, HUVEC werestimulated with rPEDF or with plPEDF. Cytosolic extracts were subjectedto immunoblotting with anti-pERK Ab (pERK, upper panel) or withanti-gERK Ab (gERK, lower panel). C and D, HUVEC were stimulated withrPEDF, plPEDF, or with the various rPEDF variants. Cytosolic extractswere subjected to immunoblotting as described above in panel A. E,Quantitative analysis of immunoblots depicted in panels C and D.

FIGS. 5A-B show the effect of rPEDF, plPEDF and the various rPEDFvariants on PEDF neurotrophic activity. Retinoblastoma cells wereincubated with rPEDF, plPEDF, or with the various rPEDF variants anddifferentiation at 10 days post-attachment is shown. B. Quantitativeanalysis of the results presented in panel A. Student t-test was used toanalyze statistical significance of the differences between cellstreated with rPEDF and cells treated with the various PEDF forms (*P<0.01, ** P<0.050).

FIGS. 6A-B show the anti-angiogenic activity of the various rPEDF formson bFGF-induced vessel sprouting in the ex-vivo aortic ring assay. A,Aortic rings were exposed to plPEDF or to the various rPEDF forms in thepresence or absence of bFGF. The rings were stained with crystal violetto illustrate sprouting and vessels formation. Micrographs were takenunder X4 and X10 objective. B. Quantitative analysis of the assaydescribed in panel A. Student t-test was used to analyze statisticalsignificance of the differences between rings treated with bFGF andrings treated with the combination of bFGF and the various PEDF forms.(* P<0.01).

FIGS. 7A-B show the anti-angiogenic activity of the various rPEDF formson bFGF-induced neovascularization in in vivo Matrigel plug assay. A,CD-1 nude mice were subcutaneously injected with Matrigel containingplPEDF or rPEDF forms in the presence or absence of bFGF. After 7 days,mice were sacrificed and Matrigel plugs were stained. Hematoxylin &Eosin staining of thin sections from Matrigel plugs are shown. B.Angiogenesis was measured by counting the number of blood vessels/fieldin each Matrigel plug. Student t-test was used to analyze statisticalsignificance of the differences between plugs treated with bFGF. andplugs treated with the combination of bFGF and the various PEDF forms (*p<0.01, ** P<0.05).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides PEDF variants, fragments and analogsthereof comprising at least one altered phosphorylation site and havinganti-angiogenic activity. The invention also provides isolated nucleicacids encoding PEDF variants, fragments and analogs thereof, the PEDFvariants, fragments and analogs thereof comprising at least one alteredphosphorylation site and having anti-angiogenic activity.

According to the present invention, the naturally occurring human PEDFof SEQ ID NO:1 contains two CK2 and one PKA phosphorylation sites. TheCK2 phosphorylation sites reside on serine residues 24 and 114, whilethe PKA phosphorylation site resides on serine residue at position 227.

According to one aspect, the present invention provides ananti-angiogenic variant of PEDF, analog, or a fusion protein thereofcomprising the amino acid sequence of SEQ ID NO:1 or a fragment thereofcomprising at least one altered phosphorylation site.

According to some embodiments, the present invention provides a PEDFvariant, fragment, analog, or a fusion protein thereof having reducedneurotrophic activity. According to some other embodiments, the PEDFvariant, fragment or analog thereof being essentially devoid ofneurotrophic activity.

According to yet other embodiments the variants of the invention retainneurotrophic activity and are useful in the treatment ofneurodegenerative disease.

The term “fragment” as used herein refers to a peptide or polypeptidecomprising only a portion of PEDF having anti-angiogenic activity. By“peptide” it is meant that the peptide comprises not more than 50 aminoacids of PEDF. By “polypeptide” it is meant that the polypeptidegenerally comprises more than 50 amino acid residues of PEDF. It will beunderstood that though the present invention relates to human PEDF,since there is high homology between human PEDF and PEDF derived fromother mammalian organisms, the present invention encompasses othermammalian PEDFs such as mouse, bovine, pig, and the like.

The term “anti-angiogenic” activity used herein is meant to define theability of PEDF to reduce or inhibit endothelial cell proliferationand/or to reduce or inhibit endothelial cell migration and/or to induceendothelial cell apoptosis, and/or to reduce or inhibitneovascularization. Anti-angiogenic activity may be detected by variousmethods known in the art. Examples of in vitro and in vivo assays forangiogenic activity include mouse corneal neovascularization, chickchorioallantoic membrane assay, rabbit corneal pocket assay, aortic ringassay, and neovascularization in Matrigel plug assay (see also examplesherein below).

The term “analog” as used herein refers to PEDF or fragments thereof,comprising altered sequences of PEDF of SEQ ID NO:1 by amino acidsubstitutions, additions, deletions, or chemical modifications. By using“amino acid substitutions”, it is meant that functionally equivalentamino acid residues are substituted for residues within the sequenceresulting in a silent change. For example, one or more amino acidresidues within the sequence can be substituted by another amino acid ofa similar polarity, which acts as a functional equivalent, resulting ina silent alteration. Substitutes for an amino acid within the sequencemay be selected from other members of the class to which the amino acidbelongs. For example, the non-polar hydrophobic) amino acids includealanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophanand methionine. The polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine. The positivelycharged (basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid. Such substitutions are known as conservativesubstitutions. Additionally, a non-conservative substitution may be madein an amino acid that does not contribute to the biological activity,e.g., anti-angiogenic activity or neurotrophic activity, of PEDF or afragment thereof. It will be appreciated that the present inventionencompasses PEDF analogs, wherein at least one amino acid is substitutedby another amino acid to produce an anti-angiogenic analog of PEDFhaving increased stability or higher half life as compared to thenaturally occurring PEDF or the wild-type recombinant PEDF.

The term “altered phosphorylation site” as used herein refers toalteration of a phosphorylation site by amino acid substitution and/orby chemical modification. It will be appreciated that substitution of aserine residue within a phosphorylation site as disclosed herein belowis meant to refer to a conservative, but preferably to anon-conservative, substitution. Thus, substitution of a serine residueresiding within a phosphorylation site such as within a CK2 or PKAphosphorylation site includes substitution to a non-polar amino acid, tonegatively charged amino acid, or to a positively charged amino acid,preferably to a negatively charged amino acid.

Since phosphorylation of a serine residue within a protein is associatedwith addition of a negatively charged phosphate group to that serine,substitution of a serine by a negatively charged amino acid is useful tocharacterize the biological significance of that phosphorylation.Importantly, while a phosphorylated protein is dephosphorylated throughthe action of phosphatases in vivo, substitution of a serine with anegatively charged amino acid yields a protein having a permanentnegatively charged amino acid at that site.

As shown herein below, substitution of the serine residue at position 24of recombinant human PEDF by a glutamic acid or substitution of bothserine residues 24 and 114 of recombinant human PEDF by glutamic acidreduced or abolished, respectively, PEDF neurotrophic activity ascompared to the neurotrophic activity of recombinant wild-type(non-mutated) PEDF. Such variants, particularly the variant havingglutamic acid at positions 24 and 114, were shown to have reduced orwere even devoid of neurotrophic activity as compared to recombinantwild-type PEDF. In addition, substitution of serine residue 227 ofrecombinant human PEDF to alanine or to glutamic acid reduced PEDFneurotrophic activity as compared to the neurotrophic activity ofrecombinant wild-type PEDF. The present invention discloses, for thefirst time, a PEDF variant, which comprises two substitutions atpositions 24 and 114 from serine to glutamic acid. The PEDF variant hassignificantly high anti-angiogenic activity and hardly any neurotrophicactivity as compared to recombinant wild-type human PEDF. It is alsodisclosed that substitution of the ser residue 227 of recombinant humanPEDF to glutamic acid reduced the anti-angiogenic activity of thevariant as compared to the anti-angiogenic activity of recombinantwild-type human PEDF.

The term “neurotrophic” activity is defined herein as the ability toinduce differentiation of a neuronal cell population. For example,PEDF's ability to induce differentiation in cultured retinoblastomacells is considered neurotrophic activity. A PEDF variant, fragment,analog or a fusion protein thereof may be essentially devoid ofneurotrophic activity. By referring to essentially devoid ofneurotrophic activity it is meant to indicate that the PEDF variant,fragment, analog or fusion protein thereof has not more than 20% of theneurotrophic activity of recombinant wild-type PEDF, preferably not morethan 10%, and more preferably not more than 5% of the neurotrophicactivity of recombinant wild-type PEDF.

The present invention encompasses PEDF analogs of which at least oneamino acid has been modified. Modifications of amino acid residuesinclude, but are not limited to, glycosylation, oxidation, permanentphosphorylation, reduction, myristylation, sulfation, acylation,acetylation, ADP-ribosylation, amidation, cyclization, disulfide bondformation, hydroxylation, iodination, methylation, derivatization byprotecting/blocking groups, or any other derivatization method known inthe art. Such alterations, which do not destroy, but may improve thePEDF activity can occur anywhere along the sequence of the PEDF variantor a fragment thereof, including at the peptide backbone, the amino acidside-chains, the amino or carboxyl termini, but preferably at aphosphorylation site. It will be appreciated that the same type ofmodification may be present in the same or varying degrees at severalsites in the protein.

The PEDF variants, PEDF fragments and analogs thereof comprising atleast one altered phosphorylation site can be produced by variousmethods known in the art, including recombinant production or syntheticproduction. Recombinant production may be achieved by the use of anisolated polynucleotide encoding a PEDF variant, fragment or analogthereof, the isolated polynucleotide operably linked to a promoter forthe expression of the polynucleotide. Optionally, a regulator of thepromoter is added. The construct comprising the polynucleotide encodingthe PEDF variant, fragment or analog thereof, the promoter, andoptionally the regulator can be placed in a vector, such as a plasmid,virus or phage vector. The vector may be used to transfect or transforma host cell, e.g., a bacterial, yeast, insect, or mammalian cell.

The present invention also encompasses PEDF fragments produced bysubjecting the PEDF variant to at least one cleavage agent. A cleavageagent may be a chemical cleavage agent, e.g., cyanogen bromide, or anenzyme, preferably an endoproteinase. Endoproteinases that can be usedto cleave the PEDF variant include trypsin, chymotrypsin, papain, V8protease or any other enzyme known in the art, which is known to produceproteolytic fragments.

Synthetic production of peptides or polypeptides is well known in theart and is available commercially from a variety of companies. A PEDFvariant, fragment or an analog thereof comprising at least one alteredphosphorylation site can be synthesized using standard direct peptidesynthesis (e.g., as summarized in Bodanszky, 1984, Principles of PeptideSynthesis (Springer-Verlag, Heidelberg), such as via solid-phasesynthesis (see, e.g., Merrifield, 1963, J. Am. Chem. Soc. 85:2149-2154).Examples of solid phase peptide synthesis methods include the BOCmethod, which utilized tert-butyloxcarbonyl as the α-amino protectinggroup, and the FMOC method, which utilizes 9-fluorenylmethyloxcarbonylto protect the α-amino of the amino acid residues, both methods arewell-known by those of skill in the art. Furthermore, if desired,non-classical amino acids or chemical amino acid analogs can beintroduced as a substitution or addition into a PEDF variant, fragmentor analog thereof. Non-classical amino acids include, but are notlimited to, oc-aminoisobutyric acid, 4-aminobutyric acid,hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine,t-butylalanine, phenylglycine, cyclohexylalanine, and the like.

The invention should further be construed to include a PEDF variant,fragment or analog thereof, which may contain one or more D-isomer formsof the amino acids of PEDF. Production of a retro-inverso D-amino acidPEDF peptide where the peptide is made with the same amino acids asdisclosed, but at least one amino acid, and perhaps all amino acids areD-amino acids is a simple matter once armed with the present invention.When all of the amino acids in the peptide are D-amino acids, and the N-and C-terminals of the molecule are reversed, the result is a moleculehaving the same structural groups being at the same positions as in theL-amino acid form of the molecule. However, the molecule is more stableto proteolytic degradation and is therefore useful in many of theapplications recited herein.

Included within the scope of the invention are chimeric, or fusionproteins comprising a PEDF variant, a fragment or analog thereof joinedat its amino or carboxy-terminus or at one of the side chains via apeptide bond to an amino acid sequence of a different protein. Suchchimeric proteins can be made by protein synthesis, e.g., by use of apeptide synthesizer, or by ligating the appropriate nucleic acidsequences encoding the desired amino acid sequences to each other bymethods known in the art, in the proper coding frame, and expressing thechimeric protein by methods commonly known in the art.

According to another aspect, the present invention provides an isolatedpolynucleotide sequence encoding a PEDF variant, a fragment, analog, ora fusion protein thereof comprising at least one altered phosphorylationsite, the PEDF variant, fragment, analog, or fusion protein thereofhaving anti-angiogenic activity. The term “PEDF variant” used throughoutthe specification and claims should be construed to include all forms ofactive PEDF variants that comprise at least one altered phosphorylationsite and having anti-angiogenic activity.

The term “polynucleotide” means a polymer of deoxyribonucleic acid (DNA)or ribonucleic acid (RNA), which can be derived from any source, can besingle- or double-stranded, and can optionally contain synthetic,non-natural, or altered nucleotides, which are capable of beingincorporated into DNA or RNA polymers.

An “isolated polynucleotide” refers to a polynucleotide segment orfragment which has been separated from sequences which flank it in anaturally occurring state, e.g., a DNA fragment which has been removedfrom the sequences which are normally adjacent to the fragment, e.g.,the sequences adjacent to the fragment in a genome in which it naturallyoccurs. The term also applies to polynucleotides, which have beensubstantially purified from other components, which naturally accompanynucleic acid, e.g., RNA or DNA or proteins, which naturally accompany itin the cell. The term therefore includes, for example, a recombinant DNAwhich is incorporated into a vector, into an autonomously replicatingplasmid or virus, or into the genomic DNA of a prokaryote or eukaryote,or which exists as a separate molecule (e.g., as a cDNA or a genomic orcDNA fragment produced by PCR or restriction enzyme digestion)independent of other sequences. It also includes a recombinant DNA,which is part of a hybrid gene encoding additional polypeptide sequence,and RNA such as mRNA.

The term “encoding” refers to the inherent property of specificsequences of nucleotides in an isolated polynucleotide, such as a gene,a cDNA, or an mRNA, to serve as templates for synthesis of otherpolymers and macromolecules in biological processes having either adefined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a definedsequence of amino acids and the biological properties resultingtherefrom. Thus, a gene encodes a protein if transcription andtranslation of mRNA corresponding to that gene produces the protein in acell or other biological system. Both the coding strand, the nucleotidesequence of which is identical to the mRNA sequence and is usuallyprovided in sequence listings, and the non-coding strand, used as thetemplate for transcription of a gene or cDNA, can be referred to asencoding the protein or other product of that gene or cDNA.

One who is skilled in the art will appreciate that more than one nucleicacid may encode any given protein in view of the degeneracy of thegenetic code and the allowance of exceptions to classical base pairingin the third position of the codon, as given by the so-called “Wobblerules.” Moreover, polynucleotides that include more or less nucleotidescan result in the same or equivalent proteins. Accordingly, it isintended that the present invention encompass all polynucleotides thatencode the amino acid sequences of SEQ ID NO:2 to SEQ ID NO:13, as wellas analog proteins. The present invention also encompassespolynucleotides with substitutions, additions, or deletions, whichdirect the synthesis of the PEDF variant, fragment, analog, or a fusionprotein thereof.

Polynucleotide sequences which encode wild type or native PEDFpolypeptides are known (see, e.g., published International PatentApplications WO 95/33480 and WO 93/24529); see also GenBank accessionno. U29953), and others can be deduced from the polypeptide sequencesdiscussed herein. According to specific embodiments, the presentinvention provides polynucleotide sequences encoding PEDF variants, thepolynucleotides selected from any one of SEQ ID NO:15 to SEQ ID NO:22.

The PEDF polynucleotides may be expressed as a transported protein wherethe PEDF variant is isolated from the medium in which the host cellcontaining the polynucleotide is grown, or may be expressed as anintracellular protein by deleting the leader or other peptides, in whichcase the PEDF is isolated from the host cells. The PEDF so isolated isthen purified by protein purification methods known in the art.

PEDF polypeptides can be provided to the tissue of interest bytransferring an expression vector comprising an isolated polynucleotideencoding a PEDF variant, fragment or analog thereof to cells associatedwith the tissue of interest. The cells produce and secrete the PEDFpolypeptide such that it is suitably provided to endothelial cellswithin the tissue to attenuate or inhibit angiogenesis within the tissueof interest. Thus, the expression vectors comprising a PEDF varianttypically include isolated polynucleotide sequences which are homologousto known PEDF sequences, e.g., they will hybridize to at least afragment of the known sequences under at least mild stringencyconditions, more preferably under moderate stringency conditions, mostpreferably under high stringency conditions (employing the definitionsof mild, moderate, and high stringency as set forth in Sambrook et al.,1989, Molecular Cloning: A Laboratory Manual, 2d edition, Cold SpringHarbor Press).

In addition to the isolated polynucleotide sequences encoding PEDFvariant polypeptides, the expression vectors comprise a promoter. In thecontext of the present invention, the promoter must be able to drive theexpression of the PEDF polynucleotide within the cells. Many viralpromoters are appropriate for use in such an expression cassette (e.g.,retroviral ITRs, LTRs, immediate early viral promoters (IEp) (such asherpes virus IEp (e.g., ICP4-IEp and ICP0-IEp) and cytomegalovirus (CMV)IEp), and other viral promoters (e.g., late viral promoters,latency-active promoters (LAPs), Rous Sarcoma Virus (RSV) promoters, andMurine Leukemia Virus (MLV) promoters). Other suitable promoters areeukaryotic promoters, which contain enhancer sequences (e.g., the rabbitβ-globin regulatory elements), constitutively active promoters (e.g.,the β-actin promoter, etc.), signal and/or tissue specific promoters(e.g., inducible and/or repressible promoters, such as a promoterresponsive to TNF or RU486, the metallothionine promoter, etc.), andtumor-specific promoters.

Within the expression vector, the polynucleotide encoding the PEDFvariant and the promoter are operably linked such that the promoter isable to drive the expression of the PEDF variant polynucleotide. As longas this operable linkage is maintained, the expression vector caninclude more than one gene, such as multiple genes separated by internalribosome entry sites (IRES). Furthermore, the expression vector canoptionally include other elements, such as splice sites, polyadenylationsequences, transcriptional regulatory elements (e.g., enhancers,silencers, etc.), or other sequences.

The expression vectors must be introduced into the cells in a mannersuch that they are capable of expressing the isolated polynucleotideencoding a PEDF variant, a fragment or analog thereof contained therein.Any suitable vector can be so employed, many of which are known in theart. Examples of such vectors include naked DNA vectors (such asoligonucleotides or plasmids), viral vectors such as adeno-associatedviral vectors (Berns et al., 1995, Ann. N.Y. Acad. Sci. 772:95-104),adenoviral vectors, herpes virus vectors (Fink et al., 1996, Ann. Rev.Neurosci. 19:265-287), packaged amplicons (Federoff et al., 1992, Proc.Natl. Acad. Sci. USA 89:1636-1640), papilloma virus vectors,picornavirus vectors, polyoma virus vectors, retroviral vectors, SV40viral vectors, vaccinia virus vectors, and other vectors. In addition tothe expression vector of interest, the vector can also include othergenetic elements, such as, for example, genes encoding a selectablemarker (e.g., β-gal or a marker conferring resistance to a toxin), apharmacologically active protein, a transcription factor, or otherbiologically active substance.

Methods for manipulating a vector comprising an isolated polynucleotideare well known in the art (see, e.g., Sambrook et al., supra) andinclude direct cloning, site specific recombination using recombinases,homologous recombination, and other suitable methods of constructing arecombinant vector. In this manner, an expression vector can beconstructed such that it can be replicated in any desired cell,expressed in any desired cell, and can even become integrated into thegenome of any desired cell.

The PEDF expression vector is introduced into the cells by any meansappropriate for the transfer of DNA into cells. Many such methods arewell-known in the art (Sambrook et al., supra; see also Watson et al.,1992, Recombinant DNA, Chapter 12, 2d edition, Scientific AmericanBooks). Thus, in the case of prokaryotic cells, vector introduction maybe accomplished, for example, by electroporation, transformation,transduction, conjugation, or mobilization. For eukaryotic cells,vectors may be introduced through the use of, for example,electroporation, transfection, infection, DNA coated microprojectiles,or protoplast fusion.

Cells into which the PEDF variant polynucleotide has been transferredunder the control of an inducible promoter if necessary, can be used astransient transformants. Such cells themselves may then be transferredinto a mammal for therapeutic benefit therein. Typically, the cells aretransferred to a site in the mammal such that the PEDF variant expressedtherein and secreted therefrom contacts the desired endothelial cells inorder that angiogenesis is inhibited. Alternatively, particularly in thecase of cells to which the vector has been added in vitro, the cells mayfirst be subjected to several rounds of clonal selection (facilitatedusually by the use of a selectable marker sequence in the vector) toselect for stable transformants. Such stable transformants are thentransferred to a mammal for therapeutic benefit therein.

The PEDF variant may also be provided to the endothelial cells bytransfecting into a population of other cells a vector comprising anisolated polynucleotide encoding a PEDF variant according to theinvention, whereby the PEDF variant is expressed in and secreted fromsaid other cells. The population of other cells so transfected is thentransferred to a site in the mammal where PEDF variant so secretedcontacts the endothelial cells and inhibits angiogenesis. Expression andsecretion of PEDF variant from the other cells then has benefit on theendothelial cells. It is not necessary that the DNA encoding PEDF bestably integrated into the cells. PEDF may be expressed and secretedfrom non-integrated or from integrated DNA in a cell.

Within the cells, the PEDF polynucleotide is expressed such that thecells express and secrete the PEDF variant polypeptide. Successfulexpression of the polynucleotide can be assessed using standardmolecular biological techniques (e.g., Northern hybridization, Westernblotting, immunoprecipitation, enzyme immunoassay, etc.). Reagents fordetecting the expression of PEDF genes and the secretion of PEDF fromtransfected cells are known in the art (see also examples herein below).

The PEDF variants produced by recombinant techniques may be purified sothat the PEDF variant will be substantially pure when administered to asubject. The term “substantially pure” refers to a compound, e.g., aprotein or polypeptide, which has been separated from components, whichnaturally accompany it. Typically, a compound is substantially pure whenat least 10%, more preferably at least 20%, more preferably at least50%, more preferably at least 60%, more preferably at least 75%, morepreferably at least 90%, and most preferably at least 99% of the totalmaterial (by volume, by wet or dry weight, or by mole percent or molefraction) in a sample is the compound of interest. Purity can bemeasured by any appropriate method, e.g., in the case of polypeptides bycolumn chromatography, gel electrophoresis or HPLC analysis. A compound,e.g., a protein, is also substantially purified when it is essentiallyfree of naturally associated components or when it is separated from thenative contaminants which accompany it in its natural state.

Pharmaceutical Compositions and Administration Routes

The present invention provides pharmaceutical compositions comprising atherapeutically effective amount of a PEDF variant, a fragment or analogthereof having anti-angiogenic activity and a pharmaceuticallyacceptable carrier, the PEDF variant, fragment or analog thereofcomprising at least one altered phosphorylation site.

The pharmaceutical compositions of the invention can be formulated asneutral or salt forms. Pharmaceutically acceptable salts include thoseformed with free amino groups such as those derived from hydrochloric,phosphoric, acetic, oxalic, tartaric acids, and the like, and thoseformed with free carboxyl groups such as those derived from sodium,potassium, ammonium, calcium, ferric hydroxides, isopropylamine,triethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

The term “pharmaceutically acceptable” means approved by a regulatoryagency of the Federal or a state government or listed in the U.S.Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the therapeuticcompound is administered. Such pharmaceutical carriers can be sterileliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil and the like, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents. Water is a preferred carrier whenthe pharmaceutical composition is administered intravenously. Salinesolutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions.Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene glycol, water, ethanol and the like. The composition, ifdesired, can also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents such as acetates, citrates or phosphates.Antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; and agents for the adjustmentof tonicity such as sodium chloride or dextrose are also envisioned.

The compositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides, microcrystallinecellulose, gum tragacanth or gelatin. Oral formulation can includestandard carriers such as pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharine, cellulose, magnesiumcarbonate, etc. Examples of suitable pharmaceutical carriers aredescribed in “Remington's Pharmaceutical Sciences” by E. W. Martin. Suchcompositions will contain a therapeutically effective amount of ananti-angiogenic PEDF variant, a fragment or analog thereof, preferablyin a substantially purified form, together with a suitable amount ofcarrier so as to provide the form for proper administration to thesubject.

The amount of the anti-angiogenic PEDF variant, a fragment or analogthereof which will be effective in the treatment of a particulardisorder or condition will depend on the nature of the disorder orcondition, and can be determined by standard clinical techniques. Inaddition, in vitro assays may optionally be employed to help identifyoptimal dosage ranges. The precise dose to be employed in theformulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.However, suitable dosage ranges for intravenous administration aregenerally about 20-500 micrograms of active compound per kilogram bodyweight. Suitable dosage ranges for intranasal administration aregenerally about 0.01 pg/kg body weight to 1 mg/kg body weight. Effectivedoses may be extrapolated from dose-response curves derived from invitro or animal model test bioassays or systems.

Depending on the location of the tissue of interest, the PEDF variantcan be supplied in any manner suitable for the provision of PEDF toendothelial cells within the tissue of interest. Thus, for example, acomposition containing a source of PEDF variant (i.e., a PEDF variantpolypeptide, or an isolated polynucleotide encoding a PEDF variant, or aPEDF variant expression vector, or cells expressing PEDF variant, asdescribed herein above) can be introduced into the systemic circulation,which will distribute the source of PEDF to the tissue of interest.Alternatively, a composition containing a source of PEDF can be appliedtopically to the tissue of interest (e.g., injected, or pumped as acontinuous infusion, or as a bolus within a tumor, applied to all or aportion of the surface of the skin, dropped onto the surface of the eye,etc.).

Methods of introduction of a pharmaceutical composition comprising asource of PEDF variant include, but are not limited to, topical,intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous,intranasal, epidural, ophthalmic, and oral routes. The compounds may beadministered by any convenient route, for example by infusion or bolusinjection, by absorption through epithelial linings (e.g., oral mucosa,rectal and intestinal mucosa, etc.), and may be administered togetherwith other therapeutically active agents. It is preferred thatadministration is localized, but it may be systemic. In addition, it maybe desirable to introduce the pharmaceutical compositions of theinvention into the central nervous system by any suitable route,including intraventricular and intrathecal injection; intraventricularinjection may be facilitated by an intraventricular catheter, forexample, attached to a reservoir. Pulmonary administration can also beemployed, e.g., by use of an inhaler or nebulizer, and formulation withan aerosolizing agent.

It may be desirable to administer the pharmaceutical composition of theinvention locally to the area in need of treatment; this may be achievedby, for example, and not by way of limitation, local infusion duringsurgery, topical application, e.g., in conjunction with a wound dressingafter surgery, by injection, by means of a catheter, by means of asuppository, or by means of an implant, said implant being of a porous,non-porous, or gelatinous material. According to some preferredembodiments, administration can be by direct injection e.g., via asyringe, at the site of a tumor or neoplastic or pre-neoplastic tissue.

For topical application, an anti-angiogenic PEDF variant can be combinedwith a pharmaceutically acceptable carrier so that an effective dosageis delivered, based on the desired activity (i.e., ranging from aneffective dosage, for example, of 1.0 pM to 1.0 mM to attenuate orprevent localized angiogenesis). In one embodiment, an anti-angiogenicPEDF variant is applied to the skin for treatment of diseases such aspsoriasis. The carrier may be in the form of, for example, and not byway of limitation, an ointment, cream, gel, paste, foam, aerosol,suppository, pad or gelled stick

A topical composition for treatment of some of the eye disorderscomprises an effective amount of an anti-angiogenic PEDF in aopthalmologically acceptable excipient such as buffered saline, mineraloil, vegetable oil such as corn or arachis oil, petroleum jelly, andMiglyol 182, alcohol solutions, or liposomes or liposome-like products.These compositions may also include preservatives, antioxidants,antibiotics, immunosuppressants, and other therapeutically effectiveagents, which do not exert a detrimental effect on the anti-angiogenicPEDF variant.

For directed internal topical applications, the pharmaceuticalcomposition may be in the form of tablets or capsules, which can containany of the following ingredients, or compounds of a similar nature: abinder such as microcrystalline cellulose, gum tragacanth or gelatin; anexcipient such as starch or lactose; a disintegrating agent such asalginic acid, Primogel, or corn starch; a lubricant such as magnesiumstearate or Sterotes; or a glidant such as colloidal silicon dioxide.When the dosage unit form is a capsule, it can contain, in addition tomaterials of the above type, a liquid carrier such as a fatty oil. Inaddition, dosage unit forms can contain various other materials whichmodify the physical form of the dosage unit, for example, coatings ofsugar, shellac, or other enteric agents.

An anti-angiogenic PEDF variant, a fragment or analog thereof can bedelivered in a controlled release system. In one embodiment, an infusionpump may be used to administer an anti-angiogenic PEDF variant, afragment or analog thereof, such as for example, that is used fordelivering insulin or chemotherapy to specific organs or tumors (seeBuchwald et al., 1980, Surgery 88: 507; Saudek et al., 1989, N. Engl. J.Med. 321: 574). In a preferred form, an anti-angiogenic PEDF variant isadministered in combination with a biodegradable, biocompatiblepolymeric implant, which releases the anti-angiogenic PEDF variant overa controlled period of time at a selected site. Examples of preferredpolymeric materials include polyanhydrides, polyorthoesters,polyglycolic acid, polylactic acid, polyethylene vinyl acetate,copolymers and blends thereof (See, Medical applications of controlledrelease, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Fla.). Inyet another embodiment, a controlled release system can be placed inproximity of the therapeutic target, thus requiring only a fraction ofthe systemic dose.

Uses of PEDF

The present invention provides a method for treating diseases ordisorders, particularly diseases or disorders associated withneovascularization. The method of treatment comprises administering to apatient in need thereof a pharmaceutical composition comprising as anactive ingredient a therapeutically effective amount of a PEDF sourceand a pharmaceutically acceptable carrier. The PEDF source according tothe present invention includes a PEDF polypeptide, e.g., theanti-angiogenic PEDF variant, a fragment, analog, or a fusion proteinthereof; an isolated polynucleotide sequence encoding the PEDFpolypeptides of the invention; an expression vector comprising theisolated polynucleotide sequence encoding the PEDF polypeptides of theinvention; and a host cell transfected with an expression vectorcomprising an isolated polynucleotide sequence encoding the PEDFpolypeptides of the invention.

The inhibition of angiogenesis is generally considered to be the haltingof the development of new blood vessels, whether they develop bysprouting or by the arrival and subsequent differentiation intoendothelial cells of circulating stem cells. However, since PEDF caninduce apoptosis of activated endothelial cells, inhibition ofangiogenesis in the context of the present invention should also beconstrued to include the killing of cells by PEDF, particularly cells inexisting vessels near or within a tumor. Thus, within the context of thepresent invention, inhibition of angiogenesis should be construed toinclude inhibition of the development of new vessels, which inhibitionmay or may not be accompanied by the destruction of nearby existingvessels. The terms “neovascularization” and angiogenesis are usedinterchangeably throughout the specification and claims.

A “therapeutic” treatment is a treatment administered to a subject whoexhibits signs of pathology for the purpose of diminishing oreliminating those signs.

A “therapeutically effective amount” of a compound is that amount ofcompound which is sufficient to provide a beneficial effect to thesubject to which the compound is administered.

Patients in need thereof may suffer from one or more disease or disorderassociated with neovascularization or may have been determined to have agreater susceptibility to a disease or disorder associated withneovascularization. Thus, the method of treatment according to thepresent invention includes both therapeutic and prophylactic utility.

Neovascular diseases and disorders that can be treated withanti-angiogenic PEDF include malignant and metastatic conditionsincluding, but not limited to, solid tumors such as sarcoma, carcinoma,fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumorleiomydsarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinoma,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma bile duct carcinoma, choriocarcinoma, seminoma,embryonal carcinoma, Wilms' tumor cervical cancer, testicular tumor,lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelialcarcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,ependymoma, Kaposi's sarcoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, andretinoblastoma.

Ocular disorders associated with neovascularization which can be treatedwith an anti-angiogenic PEDF variant, a fragment or analog thereofinclude, but are not limited to, neovascular glaucoma, diabeticretinopathy, retinoblastoma, retrolental fibroplasias, uveitis,retinopathy of prematurity, macular degeneration, corneal graftneovascularization as well as other eye inflammatory diseases, oculartumors such as retinal tumors and choroidal tumors, and diseasesassociated with retinal, choroidal or iris neovascularization.

Other disorders, which can be treated with an anti-angiogenic PEDFvariant include, but are not limited to, hemangioma, arthritis,psoriasis, angiofibroma, atherosclerotic plaques, hemophilic joints, andhypertrophic scars.

An anti-angiogenic PEDF variant can be tested in vivo for the desiredtherapeutic or prophylactic activity as well as for determination of atherapeutically effective dosage. For example, such compounds can betested in suitable animal model systems prior to testing in humans,including, but not limited to, rats, mice, chicken, cows, monkeys,rabbits, and the like. For in vivo testing, prior to administration tohumans, any animal model system known in the art may be used (seeexamples herein below).

According to another aspect, the present invention provides a method fortreating a neurodegenerative diseases or condition in a subjectcomprising administering to the subject in need thereof atherapeutically effective amount of a pharmaceutical compositionaccording to the principles of the invention and a pharmaceuticallyacceptable carrier.

“Neurotrophic” activity is defined herein as the ability to inducedifferentiation of a neuronal cell population. For example, PEDF'sability to induce differentiation in cultured retinoblastoma cells isconsidered neurotrophic activity. “Neuronotrophic” activity is definedherein as the ability to enhance survival of neuronal cell populations.For example, PEDF's ability to act as a neuron survival factor onneuronal cells is neuronotrophic activity. “Gliastatic” activity isdefined herein as the ability to inhibit glial cell growth andproliferation. For example, PEDF's ability to prevent growth and/orproliferation of glial cells is gliastatic activity.

Many neurodegenerative diseases and other insults to the CNS (brain andretina) are typified by death of neurons and overpopulation by glia(gliosis). PEDF can be used effectively in these conditions to prolongthe life and functioning of the primary neurons and to stave off theglial advance. PEDF can be effective, for example, in blockingmicroglial activation in response to CNS injury as well asprolonging/sparing the lives of neurons. In the retina, it ispredictable that PEDF inhibits the Muller glial cells. Since Mullercells are similar to astroglia, PEDF would be similarly effective inblocking gliosis in conditions such as retinal detachment, diabetes,Retinitis Pigmentosa, etc. as well as sparing the lives of the retinalneurons.

It is thought that transplantation of neurons may cure certainpathologies. For example, in Parkinson's disease, transplantation ofspecific fetal brain cells into patients could alleviate or cure theproblems associated with the disease. One of the major problems tocontend with, though, would be to prolong the life of the transplantedcells and to keep them differentiated, e.g. secreting the propersubstances, etc. Pretreatment of the cells with PEDF could aid in bothof these areas. Similarly, transfection of either neurons or astrogliawith the PEDF gene before implantation can be a long-term source of PEDFat the transplantation site.

There is much activity in attempts at transplantation of neural retinaand photoreceptor cells to help cure blindness. Attempts to date havenot been fruitful both due to non-differentiation and death of thegrafts. PEDF may help in both regards. Specifically, photoreceptorneurons to be transplanted can be pretreated with PEDF or the genetransfected into the cells before surgery. Alternatively, PEDF can betransfected at high levels into adjacent retinal pigment epithelial(RPE) cells where they can serve as a supranormal source of the protein.Several investigators have now shown that cultured RPE cells survivevery well after transplantation into the interphotoreceptor space oftest animals. Transfection of human RPE cells in vitro with the PEDFgene the use of these cells in retinal transplantation is, therefore,feasible.

Where PEDF is produced naturally, it can be present in concentrations ashigh as about 250 nM. Because PEDF variants are non-toxic, they can besupplied to tissues in a far more concentrated dosage. However, givenPEDF variant's potency, it can be employed at far reducedconcentrations, such as about 10 nM or less (e.g., as little as 0.01nM). Depending on the formulation of a composition comprising the PEDFsource, it is supplied over a time course sufficient to retardangiogenesis and/or to induce neuronal cell differentiation, i.e.,neurotrophic activity, and/or to improve neuronal cell survival, i.e.,neuronotrophic activity, and/or to inhibit glial cell proliferation,i.e, gliastatic activity within a desired tissue.

In some protocols, repeated application may enhance the anti-angiogenicactivity and/or the neurotrophic and/or neuronotrophic and/or gliastaticactivity of the PEDF variant and may be required in some applications.Where the source of PEDF is a PEDF expression vector, the cellsexpressing PEDF may produce an effective amount of the protein (i.e.,sufficient to exert one or more of the biological activities of PEDF).

PEDF variants can be administered alone or in conjunction with othertherapeutic modalities. It is appropriate to administer a PEDF variantas part of a treatment regimen involving other therapies, such assurgery, drug therapy, photodynamic therapy, and/or radiation therapy.

EXAMPLES Reagents and Antibodies

Recombinant human CK2 was purchased from Calbiochem (Darmstadt,Germany), the catalytic subunit of PKA was purified as previouslydescribed. Active ERK was purified as described. Full-length human PEDFcDNA was provided by Dr. N. Bouck (Northwestern University, Chicago,Ill., USA). Phosphothreonine Ab was purchased from Zymed Laboratories,Inc (San Francisco, Calif.). Phosphotyrosine Ab (PY99) was purchasedfrom Santa Cruz, Biotechnology (Santa Cruz, Calif.). pERK, gERKphosphoserine Abs, bFGF, α-casein and dephosphorylated casein werepurchased from Sigma (Rehovot, Israel). Polyclonal Ab against PEDF wasdeveloped by the Ab Unit of the Weizmann Institute of Science.

Cell Cultures

Human Y-79 retinoblastoma cells (ATCC, Manassa, Va.) were grown in MEMsupplemented with 2 mM L-Glutamine and 15% fetal calf serum (FCS).HEK-293T cells were cultured in DMEM F-12 supplemented with 10% FCS.HUVEC were grown in M-199 supplemented with 20% FCS, 25 μg/ml ECGSmitogen (BT-203, Biomedical Technologies Inc, Stoughton, Mass.), and 5U/ml heparin.

Construction of rPEDF Variants

Full-length PEDF cDNA was used as a template for oligonucleotide-sitedirected mutagenesis kit (Clontech, Palo Alto, Calif.). Pure PCRproducts digested by Hind III and EcoR I were ligated into themulticloning site of pcDNA3. DNA sequencing analysis confirmed thenucleotide sequence of the PEDF variants.

Transient Expression of Variants in HEK-293T Cells

pcDNA3 carrying variants were introduced into HEK-293T cells using theLipofectAMINE reagent (Life Technologies Inc, Grand Island, N.Y.)according to the manufacturer's instructions. The transfected cells wereserum starved (3 days, serum-free) after which the PEDF variants werepurified on a Ni⁺² column (Amersham, UK) according to the manufacturer'sinstructions.

Purification of PEDF from Human Plasma

plPEDF was purified from human citrated plasma (IL) by a 9-20% PEG cutfollowed by DEAE-Sephacel column (2.9×40 cm) and heparin agarose columnthat was developed stepwise. The fractions were pooled (˜20 ml),dialyzed against buffer D (20 mM Tris-HCl, pH 7.4), and applied onto aMono Q-FPLC column (1 ml, Pharmacia, Sweden), which was developed with alinear NaCl gradient in buffer D. PEDF was eluted at 0.2M NaCl andusually yielded 1 mg pure PEDF (4° C. all steps).

Alkaline Phosphatase Treatment of PEDF

Recombinant PEDF (50 μg/ml) or plPEDF (50 μg/ml) were incubated withalkaline phosphatase conjugated to acrylic beads (50 U/ml) or withsepharose CL-4B beads as control (45 min, 30° C.). Beads werepre-equilibrated with BSA (1 mg/ml), Tris-HCl (50 mM pH 8.0), and EDTA(0.1 mM). Reaction was arrested by centrifugation. The supernatant wasfurther subjected to an in vitro phosphorylation.

In vitro Phosphorylation of PEDF

The phosphorylation assay (40 μl) contained either rPEDF, plPEDF orrPEDF variants (50 μg/ml). For CK2: the constituents were CK2 (4 μg/ml),glycerol (2%), NaCl (20 mM), β-mercaptoethanol (0.1 mM), MgCl₂ (20 mM),[γ³²P]-ATP (10 μM), poly-L-lysine (200 nM), and Tris-HCl (50 mM pH 7.4).For PKA: pure catalytic subunit of PKA (2.5 μg/ml), MgCl₂ (10 mM),heparin (50 μg/ml), [γ³²P]-ATP (10 μM), and Tris-HCl (50 mM pH 6.5). Forhuman plasma: phosphatase treated PEDF (30 μg/ml), fresh human plasma,MgCl₂ (20 mM), [γ³²P]-ATP (20 μM), Tris (50 mM pH 7.4) with or withoutPKA inhibitor (PKI, 1 μg/ml) or heparin (100 μg/ml). Reactions were for45 min at 30° C. Then, boiled sample buffer was added, and the sampleswere subjected to 10% SDS-PAGE.

Determination of ERK Phosphorylation

Serum starved cells were treated with rPEDF, plPEDF or the various rPEDFvariants (10 nM unless otherwise specified) for the indicated times.Following stimulation pERK and gERK were detected using the appropriateAbs.

Neurite Outgrowth Assay

Human Y-79 retinoblastoma cells were assayed for neurite outgrowth.Briefly, one ml of a Y-79 cell suspension (2.5×10⁵ cells/ml) wasincubated with rPEDF, plPEDF or with the various rPEDF variants (20 nM)in the cell's medium. After 7 days the cells were transferred topoly-D-lysine coated plates, and their neurite outgrowth was monitoredby light microscopy at various periods of time.

Aortic Ring Assay

The aortic ring assay was performed as follows: thoracic aortas weredissected from 10-12 weeks old BALB/C mice and transferred to Petri dishcontaining BIO-MPM-1. After removing excess perivascular tissue,transverse cuts of 1 mm long were made. The rings were embedded incollagen mix (7 parts collagen, 1 part 10×MEM, and 2 parts 0.15M NaHCO₃,800111) in 24-well plates. Medium (500 μl BIO-MPM-1 containingpenicillin-streptomycin and the examined reagent) was added to theembedded rings, and the plates were incubated at 37° C. in a humidifiedincubator. Medium containing reagents was replaced 3 times a week. After10-12 days, the rings were fixed with 4% formaldehyde and stained withcrystal violet (0.02%). The effect of each factor was examined in 2wells (4 rings) per assay, and was repeated at least 3 times.

Matrigel Plug Angiogenesis Assay

Matrigel (BD Biosciences, MA; 0.5 ml/mouse) containing bFGF (300 ng/ml),with or without PEDF (20 nM) was injected subcutaneously into the flankof 8 weeks old nude mice as described by Passaniti, A. et al. (Lab.Invest. 67: 519-528, 1992). On day 7, mice were sacrificed, plugs wereremoved, fixed (4% formaldehyde), paraffin embedded and sectioned.Sections were stained using Hematoxylin-Eosin (H&E). Endothelialcells/microvessels infiltrating the Matrigel were confirmed by Masson'sTrichrome staining.

Example 1 PEDF in Plasma is a Phosphoprotein

PEDF, which was identified as a neurotrophic and antiangiogenic factorin the eye, was recently found to be present also in circulating blood.Since it was demonstrated that exokinases are able to phosphorylateplasma proteins, the experiment aimed at studying whether PEDF can be atarget for phosphorylation by these kinases. Two forms of PEDF were usedin the study: (1) PEDF purified from human plasma (plPEDF); and (2)recombinant PEDF (rPEDF), which was expressed in HEK-293T cells andpurified from the serum free medium of these cells. To examine whetherplPEDF is indeed a phosphoprotein, plPEDF and rPEDF were firstimmunoblotted with various anti-phospho amino acid Abs. Both proteinswere specifically recognized by anti-phospho-Ser Ab, but not byanti-phospho-Thr, or by anti-phospho-Tyr Abs (FIG. 1A). As positivecontrols, active phosphorylated ERK (pERK), which was recognized both byanti-phospho-Tyr and anti-phospho-Thr, and α casein, which wasrecognized only by anti-phospho-Ser Ab, were used. The results indicatedthat plPEDF and rPEDF are phosphorylated on Ser residue(s).

The existence of extracellular PKA and CK2 activities is welldocumented. Analysis of the primary amino acid sequence of PEDF revealedthe existence of several putative phosphorylation sites for CK2, as wellas for PKA. In order to examine whether PEDF can be phosphorylated byone of these protein kinases, rPEDF and plPEDF were pretreated withimmobilized alkaline phosphatase prior to an in vitro phosphorylationreaction by CK2 and PKA. Phosphorylated products were subjected toSDS-PAGE followed by Western blotting, and the membranes were firstexposed to autoradiography and then immunoblotted with anti-PEDF Ab.Pretreatment of plPEDF with alkaline phosphatase (FIG. 1B) significantlyincreased CK2, and to a lesser extent PKA phosphorylation of theprotein. The PKA and CK2 phosphorylation of rPEDF following phosphatasetreatment were also increased, but not as significantly as plPEDF (FIG.1C).

To further verify that CK2 phosphorylation of PEDF can occur in plasma,plPEDF was pretreated with alkaline phosphatase following itsphosphorylation by fresh human plasma. A phosphorylated product thatcorresponds exactly to PEDF was detected by the autoradiography (FIG. 1Dleft panel). Heparin, which is an inhibitor of CK2, and PKI, whichinhibits PKA, inhibited this reaction (FIG. 1D, right panel). Takentogether, our results indicate that PEDF is phosphorylated in thecirculating blood on the CK2 sites. The small amount of phosphorylationin the secreted rPEDF may be a result of cellular phosphorylation.

Example 2 CK2 and PKA Phosphorylate PEDF in Vitro

As plPEDF is found to be a phosphoprotein that can be phosphorylated byCK2 and PKA, the phosphorylation of PlPEDF by these kinases was nextanalyzed. Thus, rPEDF and plPEDF were incubated with CK2 and [γ³²P]-ATP,with an increasing concentration of poly-L-lysine, which activates CK2in vitro. Both rPEDF and plPEDF were phosphorylated by CK2 (FIG. 2A),and as reported for calmodulin, the phosphorylation of PEDF wasdependent on the presence of poly-L-lysine. Additionally, CK2phosphorylation of rPEDF was stronger than the phosphorylation of plPEDF(FIG. 2A), indicating that some of the plPEDF CK2 phosphorylation sitesare already phosphorylated. Heparin was found to inhibit CK2phosphorylation of PEDF (FIG. 2B).

The possibility that PEDF is an in vitro substrate of PKA was alsodetermined. rPEDF and plPEDF were incubated with the pure catalyticsubunit of PKA and [γ³²P]-ATP in the presence of heparin, whichstimulates PKA phosphorylation of several substrates. Both rPEDF andplPEDF were equally phosphorylated by PKA in the presence of heparin(FIG. 2C) in a PKI-inhibited manner (not shown), indicating that bothproteins contain only a small amount of phosphate incorporated to thePKA site.

Example 3 Localization of the CK2 Phosphorylation Site(s) in PEDF

CK2 phosphorylates Ser or Thr immersed in acidic sequence withinproteins and peptides. The minimum requirement for CK2 phosphorylationis depicted by the sequence S/T-X-X-D/E. The presence of additional Aspor Glu residues at positions −3, +1, +2, +4, +5, or +7 improves thephosphorylation efficacy. By examining the primary sequence of PEDF forpotential phosphorylation sites, 11 putative sites that meet the minimalconsensus requirements were found. These are S24, S114, T121, S195,T219, T226, S227, T287, S328, S336, and T354. Of these, S24, S114, S195,T226, S227 and T287 were considered as preferred targets since theycontain additional acidic residues in the preferred positions.

In an attempt to identify the actual CK2 phosphorylation site(s) inPEDF, rPEDF was digested with trypsin. This partial digestion yieldedtwo major fragments with an apparent molecular weight of 20 kDa and 30kDa (FIG. 2D). We then phosphorylated rPEDF by CK2 and digested thephosphorylated protein with trypsin. Only the 20 kDa fragment wasphosphorylated by CK2 (FIG. 2D), indicating that the CK2 phosphorylationsite is located within the 20 kDa fragment. The fragment could not besequenced by Edman degradation since it was blocked, indicating that itis the N-terminal fragment of PEDF. The 30 kDa fragment was sequenced byEdman degradation and was found to start at amino acid Glu198. Massspectrometry revealed more peptides in the 30 kDa fragment (FIG. 2E)confirming its C-terminal position. Since the CK2 phosphorylation sitesare located within the 20 kDa fragment, it was concluded that Ser24and/or Ser114 are the sites of CK2 phosphorylation. However, because thecombined mass of the fragments is smaller than that of the full-lengthrPEDF, it is possible that an additional CK2 phosphorylated fragment,which run out of the gel, was also formed.

Example 4 Identification of the CK2 Phosphorylation Site(s) by SiteDirected Mutagenesis

To further study the CK2 phosphorylation sites in PEDF, single or doublesite variants were constructed by replacing Ser at position 24, 114 withAla (S24A, S114A and S24,114A) or with Glu (S24E, S114E and S24,114E).rPEDF and its variants were purified from the medium of the transfectedHEK-293T cells and subjected to phosphorylation by CK2. Mutation of S24Asignificantly reduced CK2 phosphorylation (FIG. 3A), while the S24Emutation reduced phosphorylation only to a moderate extent (FIG. 3A).The S114A variant significantly reduced CK2 phosphorylation, while thedouble variant S24,114A almost completely abolished this phosphorylation(FIG. 3A). It was concluded that both Ser24 and Ser114 are the mainsites for CK2 phosphorylation of PEDF. Surprisingly, both S114E andS24,114E mutations significantly increased CK2 phosphorylation comparedwith CK2 phosphorylation of rPEDF (FIG. 3A). This unexpected resultimplies that mutation of this residue to Glu probably leads to theexposure of additional potential phosphorylation sites that werenormally covered. Analysis of the three dimensional structure of PEDFrevealed that Thr121 is spatially close to Ser114 and may serve as theadditional site. However, since this site may be covered upon phosphateincorporation to Ser24 and Ser114, it is possible that Thr354 is theother phosphorylated site. This site might have been phosphorylated byCK2 but was not detected in the tryptic digest because it was includedin a small fragment that was not present on the gels. Nonetheless, ourresults indicate that PEDF is phosphorylated by CK2 mainly on residuesSer24 and Ser114.

Example 5 Identification of the PKA Phosphorylation Site by SiteDirected Mutagenesis

PKA phosphorylates Ser or Thr residues adjacent to at least twoconsecutive basic residues, depicted by the consensus sequence ofR/K-R/K-X-S/T. By examining the primary sequence of PEDF for potentialPKA phosphorylation sites, one such putative site at Ser227 was found.In order to confirm this PKA phosphorylation site in PEDF, a single sitevariant was constructed by replacing Ser227 either with Ala (S227A) orwith Glu (S227E). The rPEDF and the variants were purified as describedherein above and subjected to phosphorylation by PKA. Mutation of Ser227to Ala or to Glu completely abolished PKA phosphorylation of both rPEDFand plPEDF (FIG. 3B), indicating that this residue is indeed the PKAsite in PEDF.

A three dimensional structure analysis of the CK2 and PKAphosphorylation sites in PEDF revealed that Ser114 and Ser227 residuesare exposed and can be accessible to interact with potential kinases.Ser24 is not included in the crystal structure, however the location ofthe N-terminus is spatially converging to Ser114. Therefore, from thestructural point of view, these residues may well serve as substratecandidates for phosphorylation.

Example 6 Activation of ERK by PEDF in Endothelial Cells

The effect of PEDF and its phosphorylated forms on the signaling andphysiological responses of endothelial cells was next studied.Therefore, serum-starved endothelial cells were incubated with rPEDF orwith plPEDF, and cell lysates were analyzed for MAPKs and PKB activityusing anti-phospho Abs. PKB as well as JNK, p38MAPK or ERK5 were notsignificantly affected in any of the conditions used (not shown). On theother hand, rPEDF caused a small (×5) but reproducible activation of ERKphosphorylation in endothelial cells, whether the cells were obtainedfrom a human source (e.g., HUVEC; FIG. 4A) or from a bovine source(e.g., BAEC; not shown). The maximal activation of ERK1/2 was obtainedafter 15 min with 10 nM PEDF. Interestingly, the activation obtainedwith plPEDF was higher than that with rPEDF in HUVEC (FIG. 4B) as wellas in BAEC (not shown).

Example 7 The Effect of rPEDF Variants on ERK Activation

Because of the differences in ERK activation between plPEDF and rPEDF,ERK activation system was used to examine whether the phosphorylationvariants indeed mimic the effect of phosphorylation on PEDF activity.When used to stimulate HUVEC, the CK2 phosphorylation site variants S24Aand S24E did not have a significant effect, while S114A and S114Evariants demonstrated slightly reduced ability to stimulate ERKphosphorylation (FIG. 4C). However, significant effects were found withthe double variants, as S24,114A had a reduced effect, while S24,114Eenhanced ERK phosphorylation (FIG. 4C). These effects were even strongerthan the effects of rPEDF or plPEDF respectively. The higher activity ofS24,114E suggests that the two Glu residues indeed mimic the activity ofphosphorylated PEDF. However, plPEDF is incompletely phosphorylated incontrast to the existence of negatively charged residues at positions 24and 114 of all molecules of the S24,114E. Similarly, the activity ofS24,114A was lower than that of rPEDF suggesting that a small fractionof the rPEDF molecules is phosphorylated on Ser 24 and 114. Thus, thevariants S24,114E and S24,114A further extent thephosphorylation-dependent differences between plPEDF and rPEDF.

Differences in ERK activation were observed also with the PKA variants.Thus, S227A completely inhibited the ability of rPEDF to induce ERK1/2phosphorylation, whereas the S227E variant had only a slight inhibitoryeffect (FIG. 4D). Similar results were obtained with BAEC (not shown).These results further indicate that rPEDF is secreted as aphosphorylated protein on residue 227, in agreement with the phosphatasestudy above. Removal of the phosphate abolishes the PEDF-induced ERKphosphorylation, while Glu at this position elevated the PEDF effect.Taken together, our results indicate that the Glu or Ala variants indeedmimic the phosphorylated or non-phosphorylated forms of PEDF.

Example 8 The Effect of rPEDF Variants on its Neurotrophic Activity

It was then aimed at studying whether CK2 as well as PKA phosphorylationof PEDF can modulate its neurotrophic activity. For that end, rPEDF,plPEDF and the various variants were used to examine their ability toinduce differentiation in human retinoblastoma Y-79 cells in culture.Indeed, rPEDF and plPEDF induced neuronal differentiation (cellaggregation and neurite outgrowth) in Y-79 cells, where the effect ofrPEDF was more pronounced compared to plPEDF (FIG. 5). The CK2phosphorylation site variants S24E/S24A and S114E/S114A had only smalleffects, as they all induced neuronal differentiation of the Y-79 cells.However, much less neurite-like processes and cell aggregates wereobserved when cells were treated with the S24,114E variant. With thisvariant, the cells formed small corona-like structures but were verycompact without any sprouts projecting from the cells, and thisinhibitory effect was stronger than that of plPEDF (FIG. 5). On theother hand, cells treated with the S24,114A variant exhibit neuriteoutgrowth and big aggregates similar to rPEDF (FIG. 5). Mutation of thePKA phosphorylation site S227E revealed a different phenotype, wherecolonies were smaller, fewer and randomly spread, although theirprocesses were clearly observed. Therefore, PKA phosphorylation has alimited influence on the neurotrophic effect of PEDF while CK2phosphorylation significantly reduces this neurotrophic effect.

Example 9 The Ex-Vivo Anti-Angiogenic Activity of rPEDF Variants

To examine the effect of phosphorylation on the antiangiogenic activityof PEDF, the ex-vivo aortic ring assay in the presence of bFGF was usedas an angiogenic model. In the presence of bFGF (50 ng/ml), aortic ringsfrom BALB/C mice developed numerous vessels-like sprouts as compared tothe rings that were treated with serum free medium (FIG. 6). Asexpected, plPEDF significantly inhibited the bFGF-induced vesselformation. However, the inhibitory effect of rPEDF was less pronouncedthan that of plPEDF, as rearrangement towards vessel formation and smallnumber of vessels structure were observed when rPEDF and bFGF were addedtogether.

The anti-angiogenic activity of the PEDF variants was then examined.When incubated together with bFGF, the CK2 non-phosphorylated doublevariant, S24,114A, exhibited an antiangiogenic activity that was similarto or slightly less then that of rPEDF, where rearrangement towardsvessels could be seen, but clear vessels were not formed (FIG. 6). Onthe other hand, the CK2 phosphorylated variant, S24,114E, appeared to bea very potent antiangiogenic factor, even stronger than plPEDF, as itdid not allow any vessel formation (FIG. 6). The PKA non-phosphorylatedvariant, S227A, inhibited the bFGF-induced vessel formation similarly torPEDF, while the PKA phosphorylated variant, S227E, had lessantiangiogenic activity (FIG. 6). S227E alone was not proangiogenic andits effect on the bFGF-induced angiogenesis was reduced as compared torPEDF. It was, therefore, concluded that phosphorylation of PEDF on itsCK2 sites significantly enhanced the antiangiogenic activity of PEDF,while the phosphorylation on its PKA site may slightly reduce itsantiangiogenic activity.

Example 10 The in Vivo Anti-Angiogenic Activity of rPEDF Variants

To further assess the effect of phosphorylation on PEDF anti-angiogenicactivity in vivo the Matrigel plug assay in the presence of bFGF wasused as an angiogenic model. Thus, liquid Matrigel supplemented with thevarious treatments was injected subcutaneously into CD-1 nude mice. TheMatrigel polymerized to form a plug, which was removed after a week andanalyzed for its angiogenic response. As expected, control plugs treatedwith PBS or PEDF alone showed very little angiogenic response (FIG. 7).bFGF-impregnated plugs elicited a robust angiogenic activity, as judgedby the large number of blood vessels infiltrating into the plug (FIG.7). plPEDF significantly inhibited the bFGF-induced vessel infiltration,while the inhibitory effect of rPEDF was significantly less pronounced(FIG. 7). As was shown in the aortic ring assay, the S24,114E varianthad even stronger antiangiogenic activity relative to plPEDF, as plugstreated with this variant had very little angiogenic response (FIG. 7).In contrast, plugs treated with bFGF and S227E had much lessantiangiogenic activity reflected in many infiltrating vessels (FIG. 7).In addition plugs treated with bFGF and S24,114A variant or S227Avariant appeared similar to those treated with bFGF and rPEDF (notshown). These results further indicate that CK2 phosphorylation enhancesthe antiangiogenic activity of PEDF, while the phosphorylation on itsPKA site may reduce this activity.

Example 11 Prevention of Angiogenesis of the Anterior Chamber of the Eyeby Systemic Administration of PEDF Variants

Four rats (250 gr each) are given three intraperitoneal injections of aPEDF variant (750 μg per dose in 5 ml water containing 3.5% ethanol) atfour-day intervals. The following day the animals are anesthetized withxylazine-ketamine and angiogenesis is induced by inoculating 2 mlheparanase (30 mg/ml) into the frontal compartment of the eye in thecornea of one of the two eyes in each rat. A fourth intraperitonealinjection of 750 μg the PEDF variant is applied the next day. Twopositive control animals receive only 2 ml heparanase (30 mg/ml) intothe frontal compartment of the eye. Angiogenesis is then allowed todevelop for 5 days at which time animals are anesthetized withxylazine-ketamine, examined and photographed under a binocularmicroscope for development of blood vessels in the anterior chamber ofthe eye. In the control eye, blood vessels appear afterheparanase-induced angiogenesis, while in the eye of a PEDFvariant-treated rat blood vessels are absent. Similar protection isobtained when angiogenesis is induced in rat eyes with bFGF.

Example 12 PEDF Variants Inhibit the Growth of Metastases

Tumor growth and specifically the ability of tumors to metastasize isangiogenesis dependent. Lewis lung carcinoma metastases are treatedsystemically with PEDF variants. Animals with Lewis lung carcinomas of600-1200 mm³ tumors are sacrificed and the skin overlying the tumor iscleaned with betadine and ethanol. In a laminar flow hood, tumor tissueis excised under aseptic conditions. A suspension of tumor cells in 0.9%normal saline is made by passage of viable tumor tissue through a sieveand a series of sequentially smaller hypodermic needles of diameter 22-to 30-gauge. The final concentration is adjusted to 1×10⁷ cells/ml andthe suspension is placed on ice. After the site is cleaned with ethanol,the subcutaneous dorsa of mice in the proximal midline are injected with1×10⁶ cells in 0.1 ml of saline.

When tumors are 1500 mm³ in size, approximately 14 days after implant,the mice undergo surgical removal of the primary tumor. The incision isclosed with simple interrupted sutures. From the day of operation, micereceive daily subcutaneous injections of a PEDF variant at a dose of 0.3mg/kg/day or of saline. When the control mice become sick frommetastatic disease (typically after 13 days of treatment), all mice aresacrificed and autopsied. Lung surface metastases are counted by meansof a stereomicroscope at 4× magnification. Lung weight, which reflectstumor burden, is measured in the PEDF variant treated and in the controlmice. Further, weight loss is also measured as a means to evaluatetoxicity in any of the mice treated with PEDF variants.

It will be appreciated by persons skilled in the art that the presentinvention is not limited by what has been particularly shown anddescribed herein above. Rather the scope of the invention is defined bythe claims that follow.

1. An anti-angiogenic variant of pigment epithelium derived factor(PEDF), an analog, or a fusion protein thereof comprising an amino acidsequence of SEQ ID NO:1 or a fragment thereof comprising at least onealtered phosphorylation site.
 2. The anti-angiogenic variant of PEDF,analog, fusion protein, or a fragment thereof according to claim 1having reduced neurotrophic activity compared to recombinant wild-typePEDF.
 3. The anti-angiogenic variant of PEDF, analog, fusion protein, ora fragment thereof according to claim 1 being essentially devoid ofneurotrophic activity.
 4. The anti-angiogenic variant of PEDF, analog,fusion protein, or a fragment thereof according to claim 1, wherein theat least one altered phosphorylation site is selected from the groupconsisting of serine 24, serine 114, and serine
 227. 5. Theanti-angiogenic variant of PEDF, analog, or a fusion protein thereofaccording to claim 1 comprising an amino acid sequence selected from anyone of SEQ ID NO:2 to SEQ ID NO:13 or a fragment thereof.
 6. Theanti-angiogenic variant of PEDF, analog, fusion protein, or a fragmentthereof according to claim 4, wherein the serine residue is substituted.7. The anti-angiogenic variant of PEDF, analog, fusion protein, or afragment thereof according to claim 6, wherein the serine residue issubstituted by a negatively charged amino acid.
 8. The anti-angiogenicvariant of PEDF, fragment, analog, or fusion protein thereof accordingto claim 7, wherein the serine residue is substituted by a glutamicacid.
 9. The anti-angiogenic variant of PEDF, analog, or fusion proteinthereof according to claim 1 comprising SEQ ID NO:2 or a fragmentthereof.
 10. The anti-angiogenic variant of PEDF, analog, or fusionprotein thereof according to claim 1 comprising SEQ ID NO:5 or afragment thereof.
 11. The anti-angiogenic variant of PEDF, analog, orfusion protein thereof according to claim 1 comprising SEQ ID NO:8 or afragment thereof.
 12. The anti-angiogenic variant of PEDF, fragment,analog, or fusion protein thereof according to claim 4, wherein theserine residue is altered by a chemical modification.
 13. Theanti-angiogenic variant of PEDF, fragment, analog, or fusion proteinthereof according to claim 11, wherein the chemical modification isselected from the group consisting of glycosylation, oxidation,permanent phosphorylation, reduction, myristylation, sulfation,acylation, acetylation, ADP-ribosylation, amidation, hydroxylation,iodination, methylation, and derivatization by blocking groups.
 14. Anisolated polynucleotide sequence encoding an anti-angiogenic variant ofpigment epithelium derived factor (PEDF), an analog, or a fusion proteinthereof, the anti-angiogenic variant of PEDF, analog or fusion proteinthereof comprising the amino acid sequence of SEQ ID NO:1 or a fragmentthereof comprising at least one altered phosphorylation site.
 15. Theisolated polynucleotide sequence according to claim 14, wherein thevariant of PEDF, analog, fusion protein or a fragment thereof havingreduced neurotrophic activity compared to recombinant wild-type PEDF.16. The isolated polynucleotide sequence according to claim 14, whereinthe variant of PEDF, analog, fusion protein or a fragment thereof beingessentially devoid of neurotrophic activity.
 17. The isolatedpolynucleotide sequence according to claim 14, wherein the at least onealtered phosphorylation site is selected from the group consisting ofserine 24, serine 114, and serine
 227. 18. The isolated polynucleotidesequence according to claim 14, wherein the variant of PEDF, analog, ora fusion protein thereof comprising the amino acid sequence selectedfrom any one of SEQ ID NO:2 to SEQ ID NO:13.
 19. The isolatedpolynucleotide sequence according to claim 17, wherein the serineresidue is substituted.
 20. The isolated polynucleotide sequenceaccording to claim 19 selected from the group consisting of SEQ ID NO:15to SEQ ID NO:22.
 21. The isolated polynucleotide sequence according toclaim 19, wherein the serine residue is substituted to a negativelycharged amino acid.
 22. The isolated polynucleotide sequence accordingto claim 21, wherein the serine residue is substituted to a glutamicacid.
 23. The isolated polynucleotide sequence according to claim 22selected from the group consisting of SEQ ID NO:15, SEQ ID NO:16, andSEQ ID NO:17.
 24. An expression vector comprising an isolatedpolynucleotide sequence encoding an anti-angiogenic variant of pigmentepithelium derived factor (PEDF), analog, or a fusion protein thereof,the anti-angiogenic variant of PEDF, analog or fusion protein thereofcomprising the amino acid sequence of SEQ ID NO:1 or a fragment thereofcomprising at least one altered phosphorylation site.
 25. The expressionvector according to claim 24, wherein the variant of PEDF, fragment,analog or fusion protein thereof having reduced neurotrophic activitycompared to recombinant wild-type PEDF.
 26. The expression vectoraccording to claim 24, wherein the variant of PEDF, fragment, analog orfusion protein thereof being essentially devoid of neurotrophicactivity.
 27. The expression vector according to claim 24, wherein theat least one altered phosphorylation site is selected from the groupconsisting of serine 24, serine 114, and serine
 227. 28. The expressionvector according to claim 24, wherein the variant of PEDF, analog orfusion protein thereof comprising the amino acid sequence selected fromany one of SEQ ID NO:2 to SEQ ID NO:13.
 29. The expression vectoraccording to claim 24, wherein the polynucleotide sequence is selectedfrom any one of SEQ ID NO:15 to SEQ ID NO:22.
 30. The expression vectoraccording to claim 27, wherein the serine residue is substituted. 31.The expression vector according to claim 30, wherein the serine residueis substituted by a negatively charged amino acid.
 32. The expressionvector according to claim 31, wherein the serine residue is substitutedby a glutamic acid.
 33. The expression vector according to claim 32,wherein the polynucleotide sequence is selected from any one of SEQ IDNO:15, SEQ ID NO:16, and SEQ ID NO:17.
 34. A host cell transfected withan expression vector according to claim
 24. 35. A pharmaceuticalcomposition comprising as an active ingredient an anti-angiogenicvariant of pigment epithelium derived factor (PEDF), analog, or a fusionprotein thereof comprising the amino acid sequence of SEQ ID NO:1 or afragment thereof, the PEDF variant, analog, fusion protein or fragmentthereof comprising at least one altered phosphorylation site, and apharmaceutically acceptable carrier.
 36. The pharmaceutical compositionaccording to claim 35, wherein the PEDF variant, analog, fusion proteinor fragment thereof having reduced neurotrophic activity compared torecombinant wild-type PEDF.
 37. The pharmaceutical composition accordingto claim 35, wherein PEDF variant, analog, fusion protein or fragmentthereof being essentially devoid of neurotrophic activity.
 38. Thepharmaceutical composition according to claim 35, wherein the at leastone altered phosphorylation site is selected from the group consistingof serine 24, serine 114, and serine
 227. 39. The pharmaceuticalcomposition according to claim 38, wherein the variant of PEDF, analog,or fusion protein thereof comprising the amino acid sequence selectedfrom any one of SEQ ID NO:2 to SEQ ID NO:13 or a fragment thereof. 40.The pharmaceutical composition according to claim 38, wherein the serineresidue is substituted.
 41. The pharmaceutical composition according toclaim 40, wherein the serine residue is substituted by a negativelycharged amino acid.
 42. The pharmaceutical composition according toclaim 41, wherein the serine residue is substituted by a glutamic acid.43. The pharmaceutical composition according to claim 41, wherein thevariant of PEDF, analog, or a fusion protein thereof selected from anyone of SEQ ID NO:2, SEQ ID NO:5, and SEQ ID NO:8.
 44. The pharmaceuticalcomposition according to claim 38, wherein the serine residue beingaltered by a chemical modification.
 45. The pharmaceutical compositionaccording to claim 44, wherein the chemical modification is selectedfrom the group consisting of glycosylation, oxidation, permanentphosphorylation, reduction, myristylation, sulfation, acylation,acetylation, ADP-ribosylation, amidation, hydroxylation, iodination,methylation, and derivatization by blocking groups.
 46. A pharmaceuticalcomposition comprising as an active ingredient an isolatedpolynucleotide sequence according to claim
 14. 47. A pharmaceuticalcomposition comprising as an active ingredient an expression vectoraccording to claim
 24. 48. A pharmaceutical composition comprising as anactive ingredient a host cell according to claim
 34. 49. A method fortreating a disease or disorder associated with neovascularization in asubject comprising administering to the subject in need thereof atherapeutically effective amount of a pharmaceutical compositionaccording to claim
 35. 50. The method according to claim 49, wherein thedisease or disorder associated with neovascularization is selected fromthe group consisting of malignant and metastatic diseases, oculardisorders, and disorders treated with anti-angiogenic factors.
 51. Themethod according to claim 49, wherein the disease or disorder associatedwith neovascularization is selected from the group consisting ofsarcoma, carcinoma, fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor leiomydsarcoma, rhabdomyosarcoma,colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer,prostate cancer, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma sweat gland carcinoma, sebaceous gland carcinoma,papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma,medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,hepatoma bile duct carcinoma, choriocarcinoma, seminoma, embryonalcarcinoma, Wilms' tumor cervical cancer, testicular tumor, lungcarcinoma, small cell lung carcinoma, bladder carcinoma, epithelialcarcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,ependymoma, Kaposi's sarcoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma,retinoblastoma, neovascular glaucoma, diabetic retinopathy,retinoblastoma, retrolental fibroplasias, uveitis, retinopathy ofprematurity, macular degeneration, corneal graft neovascularization,retinal tumors, choroidal tumors, hemangioma, arthritis, psoriasis,angiofibroma, atherosclerotic plaques, hemophilic joints, andhypertrophic scars.
 52. A method for treating a neurodegenarativecondition in a subject comprising administering to the subject in needthereof a therapeutically effective amount of a pharmaceuticalcomposition according to claim
 35. 53. A method for treating a diseaseor disorder associated with neovascularization in a subject comprisingadministering to the subject in need thereof a therapeutically effectiveamount of a pharmaceutical composition according to claim
 46. 54. Amethod for treating a disease or disorder associated withneovascularization in a subject comprising administering to the subjectin need thereof a therapeutically effective amount of a pharmaceuticalcomposition according to claim
 47. 55. A method for treating a diseaseor disorder associated with neovascularization in a subject comprisingadministering to the subject in need thereof a therapeutically effectiveamount of a pharmaceutical composition according to claim
 48. 56. Amethod for treating a neurodegenarative condition in a subjectcomprising administering to the subject in need thereof atherapeutically effective amount of a pharmaceutical compositionaccording to claim
 46. 57. A method for treating a neurodegenarativecondition in a subject comprising administering to the subject in needthereof a therapeutically effective amount of a pharmaceuticalcomposition according to claim
 47. 58. A method for treating aneurodegenarative condition in a subject comprising administering to thesubject in need thereof a therapeutically effective amount of apharmaceutical composition according to claim 48.